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Aims: The purpose of this study is to establish a novel diagnosis system in early acute coronary syndrome (ACS) using probe electrospray ionization-mass spectrometry (PESI-MS) and machine learning (ML) and to validate the diagnostic accuracy. Methods: A total of 32 serum samples derived from 16 ACS patients and 16 control patients were analyzed by PESI-MS. The acquired mass spectrum dataset was subsequently analyzed by partial least squares (PLS) regression to find the relationship between the two groups. A support vector machine, an ML method, was applied to the dataset to construct the diagnostic algorithm. Results: Control and ACS groups were separated into the two clusters in the PLS plot, indicating ACS patients differed from the control in the profile of serum composition obtained by PESI-MS. The sensitivity, specificity, and accuracy of our diagnostic system were all 93.8%, and the area under the receiver operating characteristic curve showed 0.965 (95% CI: 0.84-1). Conclusion: The PESI-MS and ML-based diagnosis system are likely an optimal solution to assist physicians in ACS diagnosis with its remarkably predictive accuracy.
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We report a centrifugal microfluidic device that automatically performs sample preparation under steady-state rotation for clinical applications using mass spectrometry. The autonomous microfluidic device was designed for the control of liquid operation on centrifugal hydrokinetics (CLOCK) paradigm. The reported device was highly stable, with less than 7% variation with respect to the time of each unit operation (sample extraction, mixing, and supernatant extraction) in the preparation process. An agitation mechanism with bubbling was used to mix the sample and organic solvent in this device. We confirmed that the device effectively removed the protein aggregates from the sample, and the performance was comparable to those of conventional manual sample preparation procedures that use high-speed centrifugation. In addition, probe electrospray ionization mass spectrometry (PESI-MS) was performed to compare the device-treated and manually treated samples. The obtained PESI-MS spectra were analyzed by partial least squares discriminant analysis, and the preparation capability of the device was found to be equivalent to that of the conventional method.
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Microfluídica , Espectrometria de Massas por Ionização por Electrospray , Centrifugação , Dispositivos Lab-On-A-Chip , RotaçãoRESUMO
Primary cilia are organelles consisting of axonemal microtubules and plasma membranes, and they protrude from the cell surface to the extracellular region and function in signal sensing and transduction. The integrity of cilia, including the length and structure, is associated with signaling functions; however, factors involved in regulating the integrity of cilia have not been fully elucidated. Here, we showed that the Rab GTPase-binding protein EHBP1L1 and its newly identified interactors CD2AP and CIN85, known as adaptor proteins of actin regulators, are involved in ciliary length control. Immunofluorescence microscopy showed that EHBP1L1 and CD2AP/CIN85 are localized to the ciliary sheath. EHBP1L1 depletion caused mislocalization of CD2AP/CIN85, suggesting that CD2AP/CIN85 localization to the ciliary sheath is dependent on EHBP1L1. Additionally, we determined that EHBP1L1- and CD2AP/CIN85-depleted cells had elongated cilia. The aberrantly elongated cilia phenotype and the ciliary localization defect of CD2AP/CIN85 in EHBP1L1-depleted cells were rescued by the expression of WT EHBP1L1, although this was not observed in the CD2AP/CIN85-binding-deficient mutant, indicating that the EHBP1L1-CD2AP/CIN85 interaction is crucial for controlling ciliary length. Furthermore, EHBP1L1- and CD2AP/CIN85-depleted cells exhibited actin nucleation and branching defects around the ciliary base. Taken together, our data demonstrate that the EHBP1L1-CD2AP/CIN85 axis negatively regulates ciliary length via actin network remodeling around the basal body.
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Actinas , Proteínas de Transporte , Cílios , Actinas/metabolismo , Cílios/metabolismo , Ligação Proteica , Proteínas rab de Ligação ao GTP/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismoRESUMO
BACKGROUND: An intraductal papillary mucinous neoplasm (IPMN) is a pancreatic tumor with malignant potential. Although we anticipate a sensitive method to diagnose the malignant conversion of IPMN, an effective strategy has not yet been established. The combination of probe electrospray ionization-mass spectrometry (PESI-MS) and machine learning provides a promising solution for this purpose. METHODS: We prospectively analyzed 42 serum samples obtained from IPMN patients who underwent pancreatic resection between 2020 and 2021. Based on the postoperative pathological diagnosis, patients were classified into two groups: IPMN-low grade dysplasia (n = 17) and advanced-IPMN (n = 25). Serum samples were analyzed by PESI-MS, and the obtained mass spectral data were converted into continuous variables. These variables were used to discriminate advanced-IPMN from IPMN-low grade dysplasia by partial least square regression or support vector machine analysis. The areas under receiver operating characteristics curves were obtained to visualize the difference between the two groups. RESULTS: Partial least square regression successfully discriminated the two disease classes. From another standpoint, we selected 130 parameters from the entire dataset by PESI-MS, which were fed into the support vector machine. The diagnostic accuracy was 88.1%, and the area under the receiver operating characteristics curve was 0.924 by this method. Approximately 10 min were required to perform each method. CONCLUSION: PESI-MS combined with machine learning is an easy-to-use tool with the advantage of rapid on-site analysis. Here, we show the great potential of our system to diagnose the malignant conversion of IPMN, which would be a promising diagnostic tool in clinical settings.
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Adenocarcinoma Mucinoso , Carcinoma Ductal Pancreático , Neoplasias Intraductais Pancreáticas , Neoplasias Pancreáticas , Humanos , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/cirurgia , Carcinoma Ductal Pancreático/patologia , Neoplasias Intraductais Pancreáticas/diagnóstico , Neoplasias Intraductais Pancreáticas/cirurgia , Adenocarcinoma Mucinoso/diagnóstico , Adenocarcinoma Mucinoso/cirurgia , Adenocarcinoma Mucinoso/patologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/cirurgia , Neoplasias Pancreáticas/patologia , Espectrometria de Massas , Aprendizado de Máquina , Estudos RetrospectivosRESUMO
BACKGROUND AND AIM: Prompt differential diagnosis of liver tumors is clinically important and sometimes difficult. A new diagnostic device that combines probe electrospray ionization-mass spectrometry (PESI-MS) and machine learning may help provide the differential diagnosis of liver tumors. METHODS: We evaluated the diagnostic accuracy of this new PESI-MS device using tissues obtained and stored from previous surgically resected specimens. The following cancer tissues (with collection dates): hepatocellular carcinoma (HCC, 2016-2019), intrahepatic cholangiocellular carcinoma (ICC, 2014-2019), and colorectal liver metastasis (CRLM, 2014-2019) from patients who underwent hepatic resection were considered for use in this study. Non-cancerous liver tissues (NL) taken from CRLM cases were also incorporated into the analysis. Each mass spectrum provided by PESI-MS was tested using support vector machine, a type of machine learning, to evaluate the discriminatory ability of the device. RESULTS: In this study, we used samples from 91 of 139 patients with HCC, all 24 ICC samples, and 103 of 202 CRLM samples; 80 NL from CRLM cases were also used. Each mass spectrum was obtained by PESI-MS in a few minutes and was evaluated by machine learning. The sensitivity, specificity, and diagnostic accuracy of the PESI-MS device for discriminating HCC, ICC, and CRLM from among a mix of all three tumors and from NL were 98.9%, 98.1%, and 98.3%; 87.5%, 93.1%, and 92.6%; and 99.0%, 97.9%, and 98.3%, respectively. CONCLUSION: This study demonstrated that PESI-MS and machine learning could discriminate liver tumors accurately and rapidly.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Espectrometria de Massas por Ionização por Electrospray/métodos , Neoplasias Hepáticas/diagnóstico , Carcinoma Hepatocelular/diagnóstico , Aprendizado de MáquinaRESUMO
BACKGROUND AND AIM: Preoperative histological evaluation of pancreatic neoplasms is important for guiding the resection strategy and preventing postoperative adverse events. However, conventional endoscopic methods have technical limitations that reduce the accuracy of the histopathological examination. Probe electrospray ionization mass spectrometry (PESI-MS) may be a useful technique for rapidly evaluating small specimens. METHODS: This single-center prospective study included patients with pancreatic neoplasms between October 2018 and December 2019. Pancreatic ductal adenocarcinoma (PDAC) specimens were obtained via endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) and non-neoplastic tissue was obtained via surgery. Specimens were subjected to PESI-MS and the mass spectra were analyzed using partial least squares regression-discriminant analysis. RESULTS: The study included 40 patients with 20 nonneoplastic specimens and 19 PDAC specimens (1 case of neuroendocrine carcinoma was omitted). All nonneoplastic specimens were sufficient for PESI-MS analysis, although only 7 of 19 PDAC specimens were sufficient for PESI-MS analysis because of poor sample quality or insufficient quantity (<1 mg). Among the 27 analyzed cases, the mass spectra clearly differentiated between the PDAC and nonneoplastic specimens. CONCLUSIONS: This study revealed that PESI-MS could differentiate between PDAC and nonneoplastic specimens, even in cases where EUS-FNA produced very small specimens.
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Biomarkers may be of value for the early detection of gastric cancer (GC) and the preoperative identification of tumor characteristics to guide treatment strategies. The present study analyzed the expression levels of phospholipids in plasma from patients with GC using liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) to detect reliable biomarkers for GC. Furthermore, combining the results with a machine learning strategy, the present study attempted to establish a diagnostic system for GC. A total of 20 plasma samples from preoperative patients with GC and 16 plasma samples from tumor-free patients (controls) were selected from our biobank named 'SHINGEN (Yamanashi Biobank of Gastroenterological Cancers)', which includes a total of 1,592 plasma samples, and were analyzed by LC/ESI-MS. The obtained data were discriminated using a machine learning-based diagnostic algorithm, whose discriminant ability was confirmed through leave-one-out cross-validation. Using LC/ESI-MS, the levels of 236 lipid molecules were determined. Biomarker analysis revealed that a few lipids that were downregulated in the GC group could discriminate between the GC and control groups. Whole lipid composition analysis using partial least squares regression revealed good discrimination ability between the GC and control groups. Integrative analysis of all molecules using the aforementioned machine learning method exhibited a diagnostic accuracy of 94.4% (specificity, 93.8%; sensitivity, 95.0%). In conclusion, the outcomes of the present study suggested the potential future application of the aforementioned system in clinical settings. By accumulating more reliable data, the present system will be able to detect early-stage cancer and will be capable of predicting the efficacy of each therapeutic strategy.
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Phytoestrogens are herbal polyphenolic compounds that exert various estrogen-like effects in animals and can be taken in easily from a foodstuff in daily life. The fallopian tube lumen, where transportation of the oocyte occurs, is lined with secretory cells and multi-ciliated epithelial cells. Recently, we showed that estrogen induces multi-ciliogenesis in the porcine fallopian tube epithelial cells (FTECs) through the activation of the estrogen receptor beta (ERß) pathway and simultaneous inhibition of the Notch pathway. Thus, ingested phytoestrogens may induce FTEC ciliogenesis and thereby affect the fecundity. To address this issue, we added isoflavones (genistein, daidzein, or glycitin) and coumestan (coumestrol) to primary culture FTECs under air-liquid interface conditions and assessed the effects of each compound. All phytoestrogens except glycitin induced multi-ciliated cell differentiation, which followed Notch signal downregulation. On the contrary, the differentiation of secretory cells decreased slightly. Furthermore, genistein and daidzein had a slight effect on the proportion of proliferating cells exhibited by Ki67 expression. Ciliated-cell differentiation is inhibited by the ERß antagonist, PHTPP. Thus, this study suggests that phytoestrogens can improve the fallopian tube epithelial sheet homeostasis by facilitating the genesis of multi-ciliated cells and this effect depends on the ERß-mediated pathway.
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Epitélio/crescimento & desenvolvimento , Receptor beta de Estrogênio/genética , Fitoestrógenos/farmacologia , Polifenóis/farmacologia , Animais , Biomimética , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Tubas Uterinas/efeitos dos fármacos , Tubas Uterinas/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , SuínosRESUMO
Background: Most pancreatic cancers are found at progressive stages when they cannot be surgically removed. Therefore, a highly accurate early detection method is urgently needed. Methods: This study analyzed serum from Japanese patients who suffered from pancreatic ductal adenocarcinoma (PDAC) and aimed to establish a PDAC-diagnostic system with metabolites in serum. Two groups of metabolites, primary metabolites (PM) and phospholipids (PL), were analyzed using liquid chromatography/electrospray ionization mass spectrometry. A support vector machine was employed to establish a machine learning-based diagnostic algorithm. Results: Integrating PM and PL databases improved cancer diagnostic accuracy and the area under the receiver operating characteristic curve. It was more effective than the algorithm based on either PM or PL database, or single metabolites as a biomarker. Subsequently, 36 statistically significant metabolites were fed into the algorithm as a collective biomarker, which improved results by accomplishing 97.4% and was further validated by additional serum. Interestingly, specific clusters of metabolites from patients with preoperative neoadjuvant chemotherapy (NAC) showed different patterns from those without NAC and were somewhat comparable to those of the control. Conclusion: We propose an efficient screening system for PDAC with high accuracy by liquid biopsy and potential biomarkers useful for assessing NAC performance.
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A moving string sampling probe and a new ESI based ionization source that can be readily incorporated into the existing endoscopes are developed for performing in vivo mass spectrometry during the endoscopic procedure. The medical-grade silk suture driven by a stepping motor is used to perform the sampling on the region of interest when the probe head is brought gently into contact with the surface of the gastrointestinal tissue. The tissues and the compounds adhered to the sampling string are transported to an ionization region inside the ion inlet tube in which they are extracted and ionized by the charging droplets generated from an electrospray outside the ion inlet. Since the extraction/ionization and sampling processes are isolated, organic solvents, high voltage (HV), and heating can be used for the optimization of ionization without compromising the biocompatibility of the sampling probe. The demonstration of the in vivo analysis of the gastric mucosa of a mouse is performed using a 2 m long gastrointestinal endoscope.
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In the developing brain, the polarity of neural progenitor cells, termed radial glial cells (RGCs), is important for neurogenesis. Intercellular adhesions, termed apical junctional complexes (AJCs), at the apical surface between RGCs are necessary for cell polarization. However, the mechanism by which AJCs are established remains unclear. Here, we show that a SNARE complex composed of SNAP23, VAMP8, and Syntaxin1B has crucial roles in AJC formation and RGC polarization. Central nervous system (CNS)-specific ablation of SNAP23 (NcKO) results in mice with severe hypoplasia of the neocortex and no hippocampus or cerebellum. In the developing NcKO brain, RGCs lose their polarity following the disruption of AJCs and exhibit reduced proliferation, increased differentiation, and increased apoptosis. SNAP23 and its partner SNAREs, VAMP8 and Syntaxin1B, are important for the localization of an AJC protein, N-cadherin, to the apical plasma membrane of RGCs. Altogether, SNARE-mediated localization of N-cadherin is essential for AJC formation and RGC polarization during brain development.
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Encéfalo/patologia , Polaridade Celular , Neuroglia/metabolismo , Neuroglia/patologia , Proteínas Qb-SNARE/deficiência , Proteínas Qc-SNARE/deficiência , Animais , Apoptose , Encéfalo/fisiopatologia , Células COS , Caderinas/metabolismo , Diferenciação Celular , Membrana Celular/metabolismo , Movimento Celular , Núcleo Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Regulação para Baixo , Marcha , Camundongos Knockout , Neurogênese , Neurônios/patologia , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE , Receptores Notch/metabolismo , Transdução de Sinais , Sintaxina 1/metabolismo , Vesículas Transportadoras/metabolismo , beta Catenina/metabolismoRESUMO
The fallopian tube (FT) is an important reproductive organ in females. The luminal epithelium of the FT is composed of highly polarized secretory and ciliated cells. Recently, accumulating lines of evidence have suggested that the origin of high-grade serous ovarian carcinoma (HGSC) is fallopian tube epithelial cells (FTECs). Due to the lack of a high-fidelity model for FTECs in vitro, homeostasis, differentiation, as well as the transformation of FTECs are still enigmatic. In this study, we optimized the culture condition for the stable expansion of basal stem cells, as well as inducing differentiation of basal cells into polarized secretory and ciliated cells in the air-liquid interface (ALI) condition suitable for long-term culture. This storable culture method of FTECs provides a versatile platform for studying differentiation mechanisms, intercellular communication, and transformation to HGSC, as well as the physiological function of the FT in vitro.
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Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Epitélio/crescimento & desenvolvimento , Tubas Uterinas/crescimento & desenvolvimento , Células-Tronco/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Separação Celular , Criopreservação , Meios de Cultura , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular , Cultura Primária de Células , SuínosRESUMO
The lumen of the fallopian tube (FT) is lined with columnar epithelium composed of secretory and ciliated cells, both of which are important for reproduction. However, the molecular mechanism regulating cell fate remains controversial. In this study, we established a primary culture system using porcine fallopian tube epithelial cells (FTECs) to study the differentiation mechanism. We found that estrogen promoted the differentiation of multi-ciliated cells (MCCs) through estrogen receptor ß, following the reduction of DLL1, a ligand of Notch. Meanwhile, epidermal growth factor (EGF), a regulator of epithelial homeostasis and differentiation, suppressed ciliogenesis by the activation of Notch signaling. However, the estrogen pathway did not affect the activation of the EGF pathway. Taken together, the differentiation of MMCs in FT depends on the balance of EGF and estrogen signaling, either of which inhibits or stimulates the Notch signaling pathway respectively.
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Cílios/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais/metabolismo , Estrogênios/metabolismo , Tubas Uterinas/metabolismo , Suínos/metabolismo , Animais , Diferenciação Celular , Células Epiteliais/citologia , Epitélio/metabolismo , Receptor beta de Estrogênio/metabolismo , Tubas Uterinas/citologia , Feminino , Receptores Notch/metabolismoRESUMO
BIG1, an activator protein of the small GTPase, Arf, and encoded by the Arfgef1 gene, is one of candidate genes for epileptic encephalopathy. To know the involvement of BIG1 in epileptic encephalopathy, we analyzed BIG1-deficient mice and found that BIG1 regulates neurite outgrowth and brain development in vitro and in vivo. The loss of BIG1 decreased the size of the neocortex and hippocampus. In BIG1-deficient mice, the neuronal progenitor cells (NPCs) and the interneurons were unaffected. However, Tbr1+ and Ctip2+ deep layer (DL) neurons showed spatial-temporal dependent apoptosis. This apoptosis gradually progressed from the piriform cortex (PIR), peaked in the neocortex, and then progressed into the hippocampus from embryonic day 13.5 (E13.5) to E17.5. The upper layer (UL) and DL order in the neocortex was maintained in BIG1-deficient mice, but the excitatory neurons tended to accumulate before their destination layers. Further pulse-chase migration assay showed that the migration defect was non-cell autonomous and secondary to the progression of apoptosis into the BIG1-deficient neocortex after E15.5. In BIG1-deficient mice, we observed an ectopic projection of corticothalamic axons from the primary somatosensory cortex (S1) into the dorsal lateral geniculate nucleus (dLGN). The thalamocortical axons were unable to cross the diencephalon-telencephalon boundary (DTB). In vitro, BIG1-deficient neurons showed a delay in neuronal polarization. BIG1-deficient neurons were also hypersensitive to low dose glutamate (5 µM), and died via apoptosis. This study showed the role of BIG1 in the survival of DL neurons in developing embryonic brain and in the generation of neuronal polarity.
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Axônios/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Interneurônios/metabolismo , Neocórtex/metabolismo , Tálamo/metabolismo , Animais , Apoptose/fisiologia , Hipocampo/metabolismo , CamundongosRESUMO
The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain-binding protein 1-like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1-dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Polaridade Celular , Células Epiteliais/enzimologia , Mucosa Intestinal/enzimologia , Intestino Delgado/enzimologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Vesículas Transportadoras/enzimologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Transporte Biológico , Proteínas de Transporte/genética , Dinaminas/metabolismo , Células Epiteliais/ultraestrutura , Células HEK293 , Células HeLa , Humanos , Mucosa Intestinal/ultraestrutura , Intestino Delgado/ultraestrutura , Lisossomos/enzimologia , Camundongos , Camundongos Knockout , Microvilosidades/enzimologia , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Organoides , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Interferência de RNA , Transdução de Sinais , Transfecção , Proteínas Supressoras de Tumor/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
West Nile virus (WNV) particles assemble at and bud into the endoplasmic reticulum (ER) and are secreted from infected cells through the secretory pathway. However, the host factor related to these steps is not fully understood. Rab proteins, belonging to the Ras superfamily, play essential roles in regulating many aspects of vesicular trafficking. In this study, we sought to determine which Rab proteins are involved in intracellular trafficking of nascent WNV particles. RNAi analysis revealed that Rab8b plays a role in WNV particle release. We found that Rab8 and WNV antigen were colocalized in WNV-infected human neuroblastoma cells, and that WNV infection enhanced Rab8 expression in the cells. In addition, the amount of WNV particles in the supernatant of Rab8b-deficient cells was significantly decreased compared with that of wild-type cells. We also demonstrated that WNV particles accumulated in the recycling endosomes in WNV-infected cells. In summary, these results suggest that Rab8b is involved in trafficking of WNV particles from recycling endosomes to the plasma membrane.
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Endossomos/enzimologia , Vírus do Nilo Ocidental/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Transporte Biológico , Chlorocebus aethiops , Endossomos/virologia , Fibroblastos/enzimologia , Fibroblastos/virologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Knockout , Transporte Proteico , Vesículas Transportadoras/virologia , Células Vero , Proteínas Virais , Liberação de Vírus , Replicação ViralRESUMO
Neurogliaform (RELN+) and bipolar (VIP+) GABAergic interneurons of the mammalian cerebral cortex provide critical inhibition locally within the superficial layers. While these subtypes are known to originate from the embryonic caudal ganglionic eminence (CGE), the specific genetic programs that direct their positioning, maturation, and integration into the cortical network have not been elucidated. Here, we report that in mice expression of the transcription factor Prox1 is selectively maintained in postmitotic CGE-derived cortical interneuron precursors and that loss of Prox1 impairs the integration of these cells into superficial layers. Moreover, Prox1 differentially regulates the postnatal maturation of each specific subtype originating from the CGE (RELN, Calb2/VIP, and VIP). Interestingly, Prox1 promotes the maturation of CGE-derived interneuron subtypes through intrinsic differentiation programs that operate in tandem with extrinsically driven neuronal activity-dependent pathways. Thus Prox1 represents the first identified transcription factor specifically required for the embryonic and postnatal acquisition of CGE-derived cortical interneuron properties. SIGNIFICANCE STATEMENT: Despite the recognition that 30% of GABAergic cortical interneurons originate from the caudal ganglionic eminence (CGE), to date, a specific transcriptional program that selectively regulates the development of these populations has not yet been identified. Moreover, while CGE-derived interneurons display unique patterns of tangential and radial migration and preferentially populate the superficial layers of the cortex, identification of a molecular program that controls these events is lacking.Here, we demonstrate that the homeodomain transcription factor Prox1 is expressed in postmitotic CGE-derived cortical interneuron precursors and is maintained into adulthood. We found that Prox1 function is differentially required during both embryonic and postnatal stages of development to direct the migration, differentiation, circuit integration, and maintenance programs within distinct subtypes of CGE-derived interneurons.
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Córtex Cerebral/citologia , Neurônios GABAérgicos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Interneurônios/citologia , Proteínas do Tecido Nervoso/fisiologia , Neurogênese/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Biomarcadores , Calbindina 2/análise , Moléculas de Adesão Celular Neuronais/análise , Linhagem da Célula , Movimento Celular , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/patologia , Proteínas da Matriz Extracelular/análise , Neurônios GABAérgicos/metabolismo , Perfilação da Expressão Gênica , Interneurônios/classificação , Interneurônios/metabolismo , Camundongos , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Proteína Reelina , Serina Endopeptidases/análise , Proteínas Supressoras de Tumor/deficiência , Peptídeo Intestinal Vasoativo/análiseRESUMO
Mammalian protein kinase D (PKD) isoforms have been proposed to regulate diverse biological processes, including the establishment and maintenance of neuronal polarity. To investigate the function of PKD in neuronal polarization in vivo, we generated PKD knockout (KO) mice. Here, we show that the brain, particularly the hippocampus, of both PKD1 KO and PKD2 KO mice was similar to that of control animals. Neurite length in cultured PKD1 KO and PKD2 KO hippocampal neurons was similar to that of wild-type neurons. However, hippocampal neurons deficient in both PKD1 and PKD2 genes showed a reduction in axonal elongation and an increase in the percentage of neurons with multiple axons relative to control neurons. These results reveal that whereas PKD1 and PKD2 are essential for neuronal polarity, there exists a functional redundancy between the two proteins.
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Polaridade Celular , Hipocampo/enzimologia , Neurônios/enzimologia , Proteína Quinase C/metabolismo , Proteínas Quinases/metabolismo , Animais , Axônios/enzimologia , Células Cultivadas , Hipocampo/citologia , Camundongos , Camundongos Knockout , Neurônios/citologia , Proteína Quinase C/genética , Proteína Quinase D2 , Proteínas Quinases/genéticaRESUMO
The small GTPase Rab11 plays an important role in the recycling of proteins to the plasma membrane as well as in polarised transport in epithelial cells and neurons. We generated conditional knockout mice deficient in Rab11a. Rab11a-deficient mice are embryonic lethal, and brain-specific Rab11a knockout mice show no overt abnormalities in brain architecture. In contrast, intestine-specific Rab11a knockout mice begin dying approximately 1 week after birth. Apical proteins in the intestines of knockout mice accumulate in the cytoplasm and mislocalise to the basolateral plasma membrane, whereas the localisation of basolateral proteins is unaffected. Shorter microvilli and microvillus inclusion bodies are also observed in the knockout mice. Elevation of a serum starvation marker was also observed, likely caused by the mislocalisation of apical proteins and reduced nutrient uptake. In addition, Rab8a is mislocalised in Rab11a knockout mice. Conversely, Rab11a is mislocalised in Rab8a knockout mice and in a microvillus atrophy patient, which has a mutation in the myosin Vb gene. Our data show an essential role for Rab11a in the localisation of apical proteins in the intestine and demonstrate functional relationships between Rab11a, Rab8a and myosin Vb in vivo.